Location: Plant, Soil and Nutrition Research
2022 Annual Report
Objectives
Objective 1: Analyze the structure and biochemical functions of selected ALMT, MATE, aquaporin (AQP), and Nramp membrane transporters in relation to Al tolerance and mineral nutrient deficiency to develop improved adaption to acid soil environments.
Objective 2: Identify the genes and molecular pathways that modulate the expression and activity of transporters that confer Al tolerance, including interacting proteins/complexes, as well as post translational modifications.
Objective 3: Dissect the signaling networks that control and regulate resistance to low pH and Al stress in Arabidopsis for ultimate application in cereal crop improvement.
Objective 4: Analyze the genetic control and the environmental regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to acid soils focusing on Al toxicity and P deficiency.
Objective 5: Analyze differential protein expression, at the cellular level in root tips, as a function of Al exposure at acidic pH, to understand specific tissue and cell type functions.
Approach
1) Identification of structural motifs that underlie key functional transport properties in the transporters associated with Al-resistance responses. We will express structurally altered transporters in heterologous systems and evaluate changes in their functionality via electrophysiological and fluxes analysis. Selected structural variants will be expressed in transgenic Arabidopsis seedling to determine their effect on the plant Al-tolerance response. 2) Functional application of Al tolerance genes for enhancing Al tolerance in crops – case studies with NRAT1, NIP1;2 and ALMT1; will be performed evaluating the levels of Al- tolerance in transgenic tomato and wheat seedlings expressing these transporters. 3) Characterization of the SbMATE interacting protein SbMBP. We will use isothermal titration calorimetry (ITC) to characterize the binding kinetics of the Al and SbMBP protein. 4) Regulation of MATE transporters via phosphorylation. We will characterize changes in the CBL/CIPK mediated changes in the transport activity of MATE transporters expressed in Xenopus oocytes via electrophysiological analysis, upon co-expression with structurally modified CIPK and CBL proteins. Identification of the phosphorylated MATE residues will be done by nanoLC-MS/MS analysis of the MATE purified protein. 5) Physiological and genetic characterization of stop1 suppressor mutants should enable the identification of new genetic and cellular components functioning in STOP1-mediated functional networks regulating Al-resistance and proton tolerance. We will perform a physiological and molecular characterization of stop1 suppressor mutants, concurrently quantifying their Al-tolerance, the magnitude of Al-induced organic acid release, and changes in gene expression of organic acid transporters involved in mediating Al-exclusion responses. The molecular identity of the suppressor mutation will be established using next-generation sequencing. 6) Analyze the genetic control and the environmental regulation of root system architecture (RSA) and the role of variation in RSA in rice and sorghum adaptation to acid soils focusing on Al toxicity and P deficiency. Using digital imagining we will quantify changes in traits defining the root architecture of rice and sorghum in response to nutrient solutions progressively modified to mimic acid soil conditions, including, but not limited to, Al-toxicity and varying phosphorus conditions. 7) Analyze differential protein expression, at the cellular level in root tips, as a function of Al exposure at acidic pH, to understand specific tissue and cell type functions. We will develop new protein labelling approaches for LC-MS/MS proteomics, thereby allowing protein quantification from various types homogeneous root cell samples harvested using laser capture microdissection (LCM). The proteomics data obtained under the various treatment will be integrated with gene expression analysis, providing information on genes that are regulated at the transcript and/or protein levels.
Progress Report
Significant progress has been made in using known Al tolerance genes from model plant systems to enhance Al tolerance in agriculturally relevant crops (Objective 1). Wheat is an experimentally recalcitrant but economically significant organism. Knowledge obtained from the various identified physiological mechanisms (and underlying genes) underlying aluminum (Al) resistance in the model organism Arabidopsis has facilitated the identification of homologous genes in wheat. We have demonstrated that coordinated functioning between two interacting genes encoding membrane transport proteins is required to enhance higher Al resistance levels in Arabidopsis. We have identified a couple of wheat genes that are functional homologs of the two Arabidopsis Al-resistance genes. By expressing these two wheat homologous genes in heterologous systems, we have shown that the wheat genes' functional properties are analogous to that described for the Arabidopsis orthologues. We continue to investigate these transport proteins' structural and biochemical properties to understand the molecular basis determining their functional properties. Overall, this research is expected to develop molecular markers for selecting Al-resistance traits in wheat. This knowledge will ultimately facilitate breeding programs to generate crop cultivars with enhanced Al resistance and yields on acid soils.
We have made significant progress in identifying molecular pathways that modulate the expression and activity of known Al-resistant genes encoding transporters that confer Al tolerance (Objective 2). We have identified two proteins that interact with membrane transport proteins known to mediate Al-tolerance. We have conducted experiments that validate and demonstrate that these proteins physically interact, modulating the level of activity of the protein, and thereby its efficiency at mediating the tolerance response. The research is expected to reveal the cellular processes underlying the resistance of sorghum plants to Al stresses and provide guidelines for the generation of crop varieties with enhanced resistance to Al toxicity and increased yields on acidic soils.
Significant progress has been made with respect to Objective 5 due to the relaxation of COVID 19 restrictions, particularly those that involve travel and visitors. We continue to refine our protocols concerning real-time-time search MS3 (RTS-MS3) quantification and its applications to spatially resolved quantitative proteomics and have published two studies this year involving single cell-type, spatially-resolved proteomics. Data sets have been deposited in the ProteomeXchange Data Repository. The first involved the identification of Al-induced protein abundance changes in the outer layers and interior tissues of the apical meristem/cell division regions of tomato roots. The second involved the identification of heat-induced proteomic changes in the meiotic pollen mother cells of the tomato "Maxifort". These papers demonstrate that single cell-type, spatially resolved proteomics is a superior experimental strategy to the analysis of complex bulked tissue samples. The new strategy enables the detection of novel metabolic pathways that contribute to the adaptation to Al and heat stress in tomato. The results of these studies justify our adoption of this approach. However, they also highlight the need for improvements in the analytical sensitivity to reduce the number of LCM harvested cells required and the time needed to collect them. Spatially resolved, single cell-type proteomics has a broad application for quantitative proteomics analysis.
Under the auspices of this work, we have continued collaboration with Tennessee State University, one of the 1890 Universities. We have fulfilled our obligation to support the 1890 Universities and the under-represented minority groups they serve through this effort.
Accomplishments
1. Improving aluminum resistance in wheat. Aluminum (Al) toxicity is an increasing problem for wheat production due to increased soil acidity caused by widely applying nitrogen-based fertilizer. Therefore, Al resistance has been an increasingly important trait for wheat breeding and yields. Al resistance is mainly achieved by a significant homologous resistance gene in both Arabidopsis and wheat. Recently, we have identified a novel Al resistance gene in Arabidopsis, which coordinately functions with the vital gene to achieve higher resistance levels. We have identified wheat sequences homologous to the Arabidopsis' second resistance gene by searching the wheat genome sequences. We functionally tested the role of the wheat homologous gene for Al resistance in Arabidopsis. We found that the wheat genes could significantly enhance aluminum resistance of the Arabidopsis transgenic plants. Our research may result in the identification of a novel Al-resistant gene in wheat and provide guidelines for breeding wheat cultivars with enhanced Al resistance and yields on acid soils.
Review Publications
Riedelsberger, J., Miller, J.K., Valdebenito-Maturana, B., Pineros, M., Gonzales, W., Dreyer, I. 2021. Plant HKT Channels: an updated view on structure, function and gene regulation. International Journal of Molecular Sciences. 22(4):1892. https://doi.org/10.3390/ijms22041892.
Manzer, Z., Ghosh, S., Jacobs, M.L., Krishnan, S., Zipfel, W.R., Pineros, M., Kamat, N.P., Daniel, S. 2021. Cell-free synthesis of a transmembrane mechanosensitive channel protein into a hybrid-supported lpid bilayer. ACS Applied Materials and Interfaces. 4(4):3101-3112. https://doi.org/10.1021/acsabm.0c01482.
Richter, A., Powell, A.F., Mirzaei, M., Wang, L.J., Movahed, N., Miller, J.K., Pineros, M., Jander, G. 2021. Indole-3-glycerolphosphate synthase, a branchpoint for the biosynthesis of tryptophan, indole, and benzoxazinoids in maize. The Plant Journal. 106:245-257. https://doi.org/10.1111/tpj.15163.
Sheng, H., Jiang, Y., Ishka, M.R., Chia, J., Dokuchayeva, T., Kavulych, Y., Zavodna, T., Mendoza, P.N., Huang, R., Smieshka, L.M., Miller, J., Woll, A.R., Terek, O.I., Romanyuk, N.D., Pineros, M., Zho, Y., Vatamaniuk, O.K. 2021. YSL3-mediated copper distribution is required for fertility, seed size and protein accumulation in Brachypodium. Plant Physiology. 186(1):655-676. https://doi.org/10.1093/plphys/kiab054.
Santiago, F., Silva, M., Cardoso, A., Duan, Y., Guilherme, L., Liu, J., Li, L. 2020. Biochemical basis of differential selenium tolerance in arugula (Eruca sativa Mill.) and lettuce (Lactuca sativa L.). Plant Physiology and Biochemistry. 157:328-338. https://doi.org/10.1016/j.plaphy.2020.11.001.
